GnRH is a decapeptide with hormonal activity with the following amino acid structure: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2 (SEQ ID NO:22), wherein the conventional three-letter code is used and pGlu is pyroglutamic acid and Gly-NH.sub.2 is glycine amide. The MRNA of GnRH comprises the GnRH sequence and a signal sequence which is cleaved of after translation followed by cyclization of the N-terminal Gln residue to form pyro-Glu.
It is known that GnRH coupled to a carrier protein can be used to vaccinate mammals. Such a vaccination can be carried out for a variety of reasons, all connected with the natural function of the GnRH. The GnRH formed in the hypothalamus regulates the production and release of the sex hormones LH (i.e. "Luteinising Hormone") and FSH ("Follicle Stimulating Hormone") in the hypophysis. A reduction of the amount of gonadotrophic hormones in the blood results in a reduced stimulation of the gonads, which results in low levels of steroids in blood. A reduction of the blood steroid level to a level comparable to the level obtained after gonadectomy can be realized by effective immunization of the animal against GnRH.
Upon administration of GnRH or its analogue as antigen, i.e. immunogen to a patient or animal, the GnRH or its analogue acts as a vaccine and the host generates antibodies to the GnRH or its analogue which also act against the body's own GnRH. Thus, the analogue's effect will persist after the analogue itself has been metabolised or excreted. This treatment is described for various GnRH analogues or GnRH itself by A. Arimura et al. in Endocrinology 93:1092-1103 (1973); by H. M. Fraser et al. in the Journal of Endocrinology 63:399406 (1974); by S. L. Jeffcoate et al. in Immunochemistry Vol. 11, p. 75-77 (1974); by I. J. Clarke et al. in the Journal of Endocrinology 78:39-47 (1978); by L. Pique et al. in Immunochemistry Vol. 15 pages 55-60 (1978); by V. C. Stevens et al. in the American Journal of Reproductive Immunology 1:307-314 (1981); and in U.S. Pat. No. 3,963,691.
In EP 0,181,236 a description is given of an immunogenic vaccine useful as an effective contraceptive agent, as an agent to treat sexual hyperactivity, for the treatment of cancers and other conditions stimulated by sexual hormones. Said vaccine comprises a conjugate between a carrier protein and one or more nona- and decapeptides derived from GnRH. Said immunisation is reversible which is an advantage over surgical methods.
The International Patent Application WO 88/05308 proposes a method for immunoneutering mammals with a composition comprising an immunogenic protein such as bovine serum albumine, conjugated with a partial peptide of GnRH having a length of 5, 6 or 7 amino acids.
Vaxstrate, the world's first commercially available contraceptive vaccine for cattle, is described as an anti-GnRH two-dose vaccine which has been shown to prevent pregnancy in about 80% of cull cows. Said vaccine comprises use of a synthetic GnRH conjugated to ovalbumin adjuvanted into an oil-based vaccine which then stimulates immunity against GnRH. Such a vaccine should further result in a higher body score and production efficiency.
According to WO 90/11298 a more reliable vaccine than the previously described vaccines can be obtained, that is particularly suited for use in prevention of boar odour of meat. Said vaccine is based on a peptide having a GnRH tandem structure preferably conjugated to a protein such as KLH. Said peptide is initially used in combination with Complete Freund's Adjuvant (CFA), followed by a booster after 8 weeks.
In WO 88/00056 a composition is described comprising two or more different carriers individually coupled to GnRH or analogues of GnRH in amounts sufficient to elicit an immune response against GnRH. Usually a protein carrier and an adjuvant are used and one or more boosters are required.
The known vaccines as described, using GnRH or its analogues after conjugation to protein carriers, are supposed to stimulate the immune system to produce anti-GnRH antibodies which should react with GnRH to effectively reduce its concentration in the body. This technique is however not effective in preventing conception for an initial period of variable length following injection.
In WO 90/03182 a solution for this problem is given by use of a composition comprising (1) free GnRH or its analogue and (2) an immunogenic conjugate between GnRH or its analogue and a carrier protein. Free GnRH or its analogue acts to prevent conception in the mammal during the period from administration to about 6 weeks, until the GnRH antibodies formed in response to the conjugate are metabolised, generally after about 0.5-2 years. The polypeptide conjugates themselves, however, have so far been immunogenically unsatisfactory.
Procedures for the conjugation of GnRH to a polypeptide carrier, e.g. bovine or human serum albumin, or tetanus toxoid or thyroglobulin, have generally involved coupling methods resulting in a poorly defined immunogen unlikely to retain all the structural features of free GnRH in solution as considered desirable from the point of view of obtaining anti-GnRH antibodies capable of blocking functioning of GnRH in vivo. Furthermore there is a danger that the peptide is attached to the carrier through a region important for immunological recognition.
Effective immunisation of mammals using such conjugates to provide a high titer of anti-GnRH antibodies capable of significantly reducing the biological efficacy of endogenous GnRH has indeed only been achieved in the presence of an adjuvant liable to cause undesirable side effects, most commonly Freund's Complete or Incomplete Adjuvant. Freund's Complete Adjuvant interferes with the tuberculin test in cattle. In addition this adjuvant as well as Freund's Incomplete Adjuvant cause a variable amount of chronic inflammatory reaction at the site of injection.
In GB 2,196,969 a vaccine is described comprising analogues of GnRH with a short peptide extension at the C-terminus of the native amino acid sequence which has been predicted by potential energy calculations to have substantially the same conformation as native GnRH in solution and may be readily linked to a polypeptide carrier via the side chain of a cysteine or tyrosine residue provided at the C-terminus.
The vaccines as described generally require large amounts of the vaccine concomitant with severe adjuvants to obtain any antibody response. They have little or no effect on biological activity connected with GnRH. Most of the vaccines described are capable of eliciting an immune response, said immune response merely comprising the formation of antibodies against GnRH and seldom comprising effect on the biological activity of GnRH in a vaccinated mammal. Those vaccines leading to a biological effect do not result in 100% immunisation.